RNA-dependent RNA polymerase

About: RNA-dependent RNA polymerase is a research topic. Over the lifetime, 13904 publications have been published within this topic receiving 767954 citations. The topic is also known as: RdRp & RNA replicase.

Computer-assisted assignment of functional domains in the nonstructural polyprotein of hepatitis E virus: delineation of an additional group of positive-strand RNA plant and animal viruses

Abstract: Computer-assisted comparison of the nonstructural polyprotein of hepatitis E virus (HEV) with proteins of other positive-strand RNA viruses allowed the identification of the following putative functional domains: (i) RNA-dependent RNA polymerase, (ii) RNA helicase, (iii) methyltransferase, (iv) a domain of unknown function ("X" domain) flanking the papain-like protease domains in the polyproteins of animal positive-strand RNA viruses, and (v) papain-like cysteine protease domain distantly related to the putative papain-like protease of rubella virus (RubV). Comparative analysis of the polymerase and helicase sequences of positive-strand RNA viruses belonging to the so-called "alpha-like" supergroup revealed grouping between HEV, RubV, and beet necrotic yellow vein virus (BNYVV), a plant furovirus. Two additional domains have been identified: one showed significant conservation between HEV, RubV, and BNYVV, and the other showed conservation specifically between HEV and RubV. The large nonstructural proteins of HEV, RubV, and BNYVV retained similar domain organization, with the exceptions of relocation of the putative protease domain in HEV as compared to RubV and the absence of the protease and X domains in BNYVV. These observations show that HEV, RubV, and BNYVV encompass partially conserved arrays of distinctive putative functional domains, suggesting that these viruses constitute a distinct monophyletic group within the alpha-like supergroup of positive-strand RNA viruses.

3'-terminal labelling of RNA with T4 RNA ligase.

Abstract: T4 RNA LIGASE catalyses the formation of an internucleotide phosphodiester bond between an oligonucleotide donor molecule with a 5′-terminal phosphate and an oligonucleotide acceptor molecule with a 3′-terminal hydroxyl1–3. Although the minimal acceptor must be a trinucleoside diphosphate, dinucleoside pyrophosphates and mononucleoside 3′,5′-bisphosphates (pNps) are effective donors in the intermolecular reaction4–6. We demonstrate here that various high molecular weight RNA molecules are acceptors in the RNA ligase reaction even when present in very low concentrations in the reaction mixture. One immediate consequence of this observation is that a convenient method for labelling the 3′ end of RNA molecules in vitro becomes available. By using a [5′-32P]pNp as a donor and RNA as an acceptor, the product of the reaction is an RNA molecule one nucleotide longer, with a 3′-terminal phosphate and a 32P-phosphate in the last internucleotide linkage. This reaction is therefore analogous to the in vitro labelling of the 5′ termini of RNA chains with polynucleotide kinase and [γ-32P]ATP and can be used in situations where 5′ labelling is not possible. In addition, the ability to add various donors to an RNA molecule should allow the function of the 3′ terminus of the molecule to be investigated.

A Mechanism for Initiating RNA-Dependent RNA Polymerization

Abstract: In most RNA viruses, genome replication and transcription are catalysed by a viral RNA-dependent RNA polymerase. Double-stranded RNA viruses perform these operations in a capsid (the polymerase complex), using an enzyme that can read both single- and double-stranded RNA. Structures have been solved for such viral capsids, but they do not resolve the polymerase subunits in any detail. Here we show that the 2 A resolution X-ray structure of the active polymerase subunit from the double-stranded RNA bacteriophage straight phi6 is highly similar to that of the polymerase of hepatitis C virus, providing an evolutionary link between double-stranded RNA viruses and flaviviruses. By crystal soaking and co-crystallization, we determined a number of other structures, including complexes with oligonucleotide and/or nucleoside triphosphates (NTPs), that suggest a mechanism by which the incoming double-stranded RNA is opened up to feed the template through to the active site, while the substrates enter by another route. The template strand initially overshoots, locking into a specificity pocket, and then, in the presence of cognate NTPs, reverses to form the initiation complex; this process engages two NTPs, one of which acts with the carboxy-terminal domain of the protein to prime the reaction. Our results provide a working model for the initiation of replication and transcription.